The overall goal of this proposal is to better understand the multiple roles of protein phosphorylation in regulating circadian rhythms by focusing on PERIOD (PER), a core transcriptional repressor of the circadian transcriptome and the key biochemical timer that underlies animal circadian time-keeping mechanisms. In animal clocks, de novo synthesized PER goes through a dynamic multi-site phosphorylation program that is dependent on the activities of a number of kinases and phosphatases. Through evolutionary fine-tuning, completion of this phosphorylation cycle requires the duration of a circadian day, thereby closely linking PER phosphorylation program to the speed of the clock. The phase-specific phosphorylation program of PER provides sophisticated time-of-day specific modulations to its functional properties, including stability, subcellular localization, and transcriptional activity, thereby controlling the time and duration for which PER functions as a repressor of the circadian transcriptome. We have previously performed mechanistic studies on specific PER phosphorylation sites that are dependent on DOUBLETIME (DBT) and NEMO kinase activities, and found that they are integral part of a phosphorylation circuitry that sets te pace of the clock. In order to fully comprehend the role of DBT and NEMO-dependent PER phosphorylation in circadian biology, we are proposing to use Drosophila melanogaster as a model to address three main questions. (i) What are the sites that are modified by DBT and NEMO? (ii) What is the temporal progression of the phosphorylation program? (iii) What is the function of specific phosphorylation events? Our specific aims are to (1) map DBT- and NEMO-dependent PER phosphorylation sites using quantitative mass spectrometry; (2) decipher the temporal progression of the PER phosphorylation program in whole animals using phosphospecific antibodies in combination with laser scanning confocal microscopy; and (3) assay changes in PER interactome with respect to kinase activities and time using AP-MS (Affinity Purification and Mass Spectrometry) in order to generate new hypotheses regarding temporal changes in PER functional properties. By integrating the results from our three aims, we will understand how dynamic phase-specific phosphorylation of Drosophila PER progresses throughout the circadian day and how specific PER phosphorylation states impacts its role in circadian time-keeping. Results from our studies could pave the way for development of small molecule therapeutics and have profound impact on treatment of human disorders associated with phosphorylation defects, e.g. Familial Advanced Sleep Phase Syndrome (FASPS).